Project Summary/Abstract The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of cellular Ca2+- signals. CaMKII is a multifunctional protein kinase that participates in a variety of different signaling events, which intriguingly can have opposing signaling outcomes (such as cell death versus survival, proliferation versus cell cycle arrest, and potentiation versus depression of synaptic strength). However, the mechanisms that differentiate between such opposing signaling outcomes mediated by CaMKII are currently unclear. A long-proposed attractive mechanism is differential CaMKII auto-phosphorylation at T286, which renders the kinase partially ?autonomous? (i.e. Ca2+-independent). However, experimental evidence is lacking, and preliminary studies of this proposal led to the alternative hypothesis that differentiation is instead mediated by auto-phosphorylation at T305/306, which prevents Ca2+/CaM binding to the kinase. The specific signaling outcomes studied here are long-term potentiation (LTP) and depression (LTD) of synaptic strength, two opposing forms of Ca2+-dependent synaptic plasticity that are induced by high or low frequency stimulation, respectively, and are thought to underlie learning and memory. Over 22 years of research has firmly linked CaMKII to LTP regulation, while CaMKII requirement in LTD is just emerging (including by the preliminary results of this proposal). Contrary to traditional models, T286 auto-phosphorylation is efficiently induced by both LTP- and LTD-stimuli. By contrast, preliminary results indicate that T305/306 auto- phosphorylation is induced exclusively by LTD- but not LTP-stimuli. Theoretical arguments can be made for the biochemical mechanisms that may underlie such stimulus-dependent differential T305/306 autophosphorylation. However, in contrast to the well-studied T286 auto-phosphorylation, little is currently known about the actual holoenzyme mechanisms governing T305/306 phosphorylation. Another important question is how T305/306 auto-phosphorylation may then lead to the opposing down-stream consequences. Preliminary results indicate that it can cause differential CaMKII substrate selection that should indeed promote LTD and suppress LTP. Thus, this proposal will: (1) determine the holoenzyme mechanism underlying the LTD-specific induction (and LTP-specific suppression) of T305/306 phosphorylation, (2) determine the specific occurance and requirement of T305/306 phosphorylation in LTD, (3) determine the requirement for T305/306 (and T286) phosphorylation in communicating excitatory LTP- or LTD-stimuli to inhibitory synapses, where these stimuli induce plasticity in the opposite direction. The results will provide a new conceptual and mechanistic framework of how a single mediator can be required in signal transduction events with opposing outcomes. A better understanding of the specific mechanism studies here will also have impact on new therapeutic strategies for Angelman Syndrome, where a CaMKII T305/306 hyper-phosphorylation is involved in synaptic and learning dysfunctions.